Synthesis of long nucleic acid sequences

ABSTRACT

The invention provides methods for the synthesis of long oligonucleotides, genes and gene fragments. The methods include the manufacture of genes or gene fragments that can be then inserted into a variety of vectors.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is a continuation of U.S. Nonprovisional patentapplication Ser. No. 14/865,127 filed Sep. 25, 2015, which claimspriority to U.S. Nonprovisional patent application Ser. No. 13/742,959filed Jan. 16, 2013 which claims priority to U.S. Provisional PatentApplication No. 61/587,073 filed Jan. 16, 2012, the content of which isincorporated herein by reference in its entirety.

SEQUENCE LISTING

The sequence listing is filed with the application in electronic formatonly and is incorporated by reference herein. The sequence listing textfile “Gene Assembly PA2013-01 Sequence Listing” was created on Jan. 16,2013 and is 34,660 bytes in size.

FIELD OF THE INVENTION

This invention pertains to the synthesis of genes or gene fragments.

BACKGROUND OF THE INVENTION

Synthetic DNA sequences are a vital tool in molecular biology. They areused in gene therapy, vaccines, DNA libraries, environmentalengineering, diagnostics, tissue engineering and research into geneticvariants. Long artificially-made nucleic acid sequences are commonlyreferred to as synthetic genes; however the synthesized artificialelements do not have to encode genes, but, for example, can beregulatory or structural elements. Regardless of functional usage, longartificially-assembled nucleic acids will be referred to herein assynthetic genes and the process of manufacturing these species will bereferred to as gene synthesis. Gene synthesis provides an advantageousalternative from obtaining genetic elements through traditional means,such as isolation from a genomic DNA library, isolation from a cDNAlibrary, or PCR cloning. Traditional cloning requires availability of asuitable library constructed from isolated natural nucleic acids whereinthe abundance of the gene element of interest is at a level that assuresa successful isolation and recovery. Further, a gene isolated fromgenomic DNA or cDNA libraries only provides an isolate having thatnucleic acid sequence as it exists in nature. It is often desirable tointroduce alterations into that sequence. For example, gene synthesisallows for complete revision of codon usage, which may be necessary toachieve efficient synthesis and expression of a human gene product in abacterial vector. As another example, a synthetic gene can haverestriction sites removed and new sites added. As yet another example, asynthetic gene can have novel regulatory elements or processing signalsincluded which are not present in the native gene. Many other examplesof the utility of gene synthesis are well known to those with skill inthe art.

Artificial gene synthesis can also provide a DNA sequence that is codonoptimized. Given codon redundancy, many different DNA sequences canencode the same amino acid sequence. Codon preferences differ betweenorganisms and a gene sequence that is expressed well in one organismmight be expressed poorly or not at all when introduced into a differentorganism. The efficiency of expression can be adjusted by changing thenucleotide sequence so that the element is well expressed in whateverorganism is desired, e.g., it is adjusted for the codon bias of thatorganism. Widespread changes of this kind are easily made using genesynthesis methods but are not feasible using site-directed mutagenesisor other methods which introduce alterations into naturally isolatednucleic acids.

Gene synthesis employs synthetic oligonucleotides as the primarybuilding block. Oligonucleotides are typically made using chemicalsynthesis, most commonly using betacyanoethyl phosphoramidite methods,which are well-known to those with skill in the art (M. H. Caruthers,Methods in Enzymology 154, 287-313 (1987)). Using a four-step process,phosphoramidite monomers are added in a 3′ to 5′ direction to form anoligonucleotide chain. During each cycle of monomer addition, a smallamount of oligonucleotides will fail to couple (n−1 product). Therefore,with each subsequent monomer addition the cumulative population offailures grows. Also, as the oligonucleotide grows longer, the baseaddition chemistry becomes less efficient, presumably due to stericissues with chain folding. Typically, oligonucleotide synthesis proceedswith a base coupling efficiency of around 99.0 to 99.2%. A 20 base longoligonucleotide requires 19 base coupling steps. Thus assuming a 99%coupling efficiency, a 20 base oligonucleotide should have 0.99¹⁹purity, meaning approximately 82% of the final end product will be fulllength and 18% will be truncated failure products. A 40 baseoligonucleotide should have 0.99³⁹ purity, meaning approximately 68% ofthe final end product will be full length and 32% will be truncatedfailure products. A 100 base oligonucleotide should have 0.99⁹⁹ purity,meaning approximately 37% of the final product will be full length and63% will be truncated failure products. In contrast, if the efficiencyof base coupling is increased to 99.5%, then a 100 base oligonucleotideshould have a 0.995⁹⁹ purity, meaning approximately 61% of the finalproduct will be full length and 39% will be truncated failure products.

Using gene synthesis methods, a series of synthetic oligonucleotides areassembled into a longer synthetic nucleic acid, e.g. a synthetic gene.The use of synthetic oligonucleotide building blocks in gene synthesismethods with a high percentage of failure products present will decreasethe quality of the final product, requiring implementation of costly andtime-consuming error correction methods. For this reason, relativelyshort synthetic oligonucleotides in the 40-60 base length range havetypically been employed in gene synthesis methods, even though longeroligonucleotides could have significant benefits in assembly. It is wellappreciated by those with skill in the art that use of high qualitysynthetic oligonucleotides, e.g. oligonucleotides with few error ormissing bases, will result in high quality assembly of synthetic genesthat use of lower quality synthetic oligonucleotides.

Some common forms of gene assembly are ligation-based assembly,PCR-driven assembly (see Tian et al., Mol. BioSyst., 5, 714-722 (2009))and thermodynamically balanced inside-out based PCR (TBIO) (see Gao X.et al., Nucleic Acids Res. 31, e143). All three methods combine multipleshorter oligonucleotides into a single longer end-product.

Therefore, to make genes that are typically 500 to many thousands ofbases long, a large number of smaller oligonucleotides are synthesizedand combined through ligation, overlapping, etc., after synthesis.Typically, gene synthesis methods only function well when combining alimited number of synthetic oligonucleotide building blocks and verylarge genes must be constructed from smaller subunits using iterativemethods. For example, 10-20 of 40-60 base overlapping oligonucleotidesare assembled into a single 500 base subunit due to the need foroverlapping ends, and twelve or more 500 base overlapping subunits areassembled into a single 5000 base synthetic gene. Each subunit of thisprocess is typically cloned (i.e., ligated into a plasmid vector,transformed into a bacterium, expanded, and purified) and its DNAsequence is verified before proceeding to the next step. If the abovegene synthesis process has low fidelity, either due to errors introducedby low quality of the initial oligonucleotide building blocks or duringthe enzymatic steps of subunit assembly, then increasing numbers ofcloned isolates must be sequence verified to find a perfect clone tomove forward in the process or an error-containing clone must have theerror corrected using site directed mutagenesis. Regardless, sequenceerrors increase cost and increase time of the manufacturing process. Anyimprovement in quality of the input oligonucleotides or assembly methodthat increases fidelity of the final product is desirable.

The methods of the invention described herein provide high qualityoligonucleotide subunits that are ideal for gene synthesis and improvedmethods to assemble said subunits into longer genetic elements. Theseand other advantages of the invention, as well as additional inventivefeatures, will be apparent from the description of the inventionprovided herein.

BRIEF SUMMARY OF THE INVENTION

The methods include the manufacture of long double-stranded nucleic acidelements that can optionally be inserted into a variety of vectors andclonally amplified.

In one embodiment, synthetic nucleic acid elements are diluted,typically to 0.25 to 10 copies per reaction well, amplified and sequenceverified, resulting in a large amount of homogeneous, desired product (a“gene block”). In a further embodiment, the synthetic nucleic acidelements are diluted to 1-5 copies of the synthesized oligonucleotideper reaction well, and in a further embodiment the synthetic nucleicacid elements are diluted to 2-4 copies per reaction well. In a furtherembodiment, the synthetics nucleic acid elements are greater than 60bases. In a further embodiment, the synthetics nucleic acid elements aregreater than 100 bases.

In a further embodiment, the gene block is comprised of two or moresmaller synthetic nucleic acid elements (“gene sub-blocks”) that arebound or covalently linked together to form the gene block, which isthen diluted. The gene block can be joined with one or more additionalgene blocks to make longer fragments in an iterative fashion.

The gene blocks can then be inserted into vectors, such as bacterial DNAplasmids, and clonally amplified through methods well-known in the art.

In a further embodiment, gene blocks are synthesized or combined in sucha manner as to provide 3′ and 5′ flanking sequences that enable thesynthetic nucleic acid elements to be more easily inserted into a vectorfor isothermal amplification.

In another embodiment, the component oligonucleotide(s) that areemployed to synthesize the synthetic nucleic acid elements arehigh-fidelity (i.e., low error) oligonucleotides synthesized on supportscomprised of thermoplastic polymer and controlled pore glass (CPG),wherein the amount of CPG per support by percentage is between 1-8% byweight.

In another embodiment, a set of oligonucleotides are joined or combinedthrough top-strand PCR amplification (TSP), wherein a plurality ofoligonucleotides covering the entire sequence of one strand of thedesired product and have a partial sequence overlap to the adjacentoligonucleotide(s), and wherein amplification is performed withuniversal forward and reverse primers, and through amplification cyclinggradually results in full-length desired product that can then undergodilution, sequence screening, and further amplification that results inthe desired gene block end product.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an illustration of top-strand PCR gene assembly. The upperportion, A, represents the first cycle of PCR, B the second cycle, and Cis the desired full-length product.

FIG. 2A is a gel showing the successful generation of the desired geneblock assembled using six gene sub-blocks. FIG. 2B is a gel showing thesuccessful generation of the desired gene block assembled using eightgene sub-blocks.

FIGS. 3A and 3B graphically represent the amount of full length cloneswith no errors for each set of reactions.

FIG. 4 depicts the 3 gene sub-block designs used to assemble the geneblocks according to Example 2.

FIG. 5 is a collection of gels showing successful generation of thedesired gene block assembled using varying gene sub-block designs usingvarious sub-block concentrations.

FIGS. 6A and 6B graphically represent the amount of full length cloneswith no errors for each set of reactions in Example 2.

FIG. 7 is a gel showing a successful synthesis and assembly of a geneblock as described in Example 3.

FIG. 8 is a gel showing a successful synthesis and assembly of an 8sub-block gene block and a 10 sub-block gene block as described inExample 4.

DETAILED DESCRIPTION OF THE INVENTION

Aspects of this invention relate to methods for synthesis of syntheticnucleic acid elements that may comprise genes or gene fragments. Morespecifically, the methods of the invention include methods of highfidelity oligonucleotide synthesis, the methods of use of high fidelityoligonucleotide synthesis in assembly/amplification methods such astop-strand PCR, and methods of gene assembly that yield a desiredsequence, a gene block, through dilution of crude synthesized geneproduct, sequence verification and subsequent amplification.

The term “oligonucleotide,” as used herein, refer topolydeoxyribonucleotides (containing 2-deoxy-D-ribose),polyribonucleotides (containing D-ribose), and to any other type ofpolynucleotide which is an N glycoside of a purine or pyrimidine base.There is no intended distinction in length between the terms “nucleicacid”, “oligonucleotide” and “polynucleotide”, and these terms can beused interchangeably. These terms refer only to the primary structure ofthe molecule. Thus, these terms include double- and single-stranded DNA,as well as double- and single-stranded RNA, or double- andsingle-stranded oligonucleotides containing both RNA and DNA. For use inthe present invention, an oligonucleotide also can comprise nucleotideanalogs in which the base, sugar or phosphate backbone is modified aswell as non-purine or non-pyrimidine nucleotide analogs.

The terms “raw material oligonucleotide” and “gene sub-blocks” are usedinterchangeably in this application and refer to the initialoligonucleotide material that is further processed, synthesized,combined, joined, modified, transformed, purified or otherwise refinedto form the basis of another oligonucleotide product. The raw materialoligonucleotides are typically, but not necessarily, theoligonucleotides that are directly synthesized using phosphoramiditechemistry.

The oligonucleotides used in the inventive methods can be synthesizedusing any of the methods of enzymatic or chemical synthesis known in theart, although phosphoramidite chemistry is the most common. Theoligonucleotides may be synthesized on solid supports such as controlledpore glass (CPG), polystyrene beads, or membranes composed ofthermoplastic polymers that may contain CPG. Oligonucleotides can alsobe synthesized on arrays, on a parallel microscale using microfluidics(Tian et al., Mol. BioSyst., 5, 714-722 (2009)), or known technologiesthat offer combinations of both (see Jacobsen et al., U.S. Pat. App. No.2011/0172127).

Synthesis on arrays or through microfluidics offers an advantage overconventional solid support synthesis by reducing costs through lowerreagent use. The scale required for gene synthesis is low, so the scaleof oligonucleotide product synthesized from arrays or throughmicrofluidics is acceptable. However, the synthesized oligonucleotidesare of lesser quality than when using solid support synthesis (See Tianinfra.; see also Staehler et al., U.S. Pat. App. No. 2010/0216648). Highfidelity oligonucleotides are required in some embodiments of themethods of the present invention, and therefore array or microfluidicoligonucleotide synthesis will not always be compatible.

In one embodiment of the present invention, the oligonucleotides thatare used for gene synthesis methods are high-fidelity oligonucleotides(average coupling efficiency is greater than 99.2%, or more preferably99.5%). In one embodiment, the high-fidelity nucleotides are between40-200 bases long. In a further embodiment the high-fidelityoligonucleotide is between 75-200 bases, and in a further embodiment100-190 bases. High-fidelity oligonucleotides are availablecommercially, even at greater lengths (see Ultramer® oligonucleotidesfrom Integrated DNA Technologies, Inc.). Alternatively, a novel methodof the present invention is the use of low-CPG load solid supports thatprovide synthesis of high-fidelity oligonucleotides while reducingreagent use. Solid support membranes are used wherein the composition ofCPG in the membranes is no more than 8% of the membrane by weight.Membranes known in the art are typically 20-50% (see for example, Ngo etal., U.S. Pat. No. 7,691,316). In a further embodiment, the compositionof CPG in the membranes is no more than 5% of the membrane. Themembranes offer scales as low as subnanomolar scales that are ideal forthe amount of oligonucleotides used as the building blocks for genesynthesis. Less reagent amounts are necessary to perform synthesis usingthese novel membranes. The membranes can provide as low as 100-picomolescale synthesis or less. The low-CPG membranes offer higher fidelity ofarray synthesis while still allowing for lower reagent use. Lower-CPGmembranes are most practical when used to synthesize oligonucleotidesgreater than 50 bases, or further, greater than 75 bases.

Other methods are known in the art to produce high-fidelityoligonucleotides. Enzymatic synthesis or the replication of existing PCRproducts traditionally has lower error rates than chemical synthesis ofoligonucleotides due to convergent consensus within the amplifyingpopulation. However, further optimization of the phosphoramiditechemistry can achieve even greater quality oligonucleotides, whichimproves any gene synthesis method. A great number of advances have beenachieved in the traditional four-step phosphoramidite chemistry since itwas first described in the 1980's (see for example, Sierzchala, et al.J. Am. Cem. Soc., 125, 13427-13441 (2003) using peroxy aniondeprotection; Hayakawa et al., U.S. Pat. No. 6,040,439 for alternativeprotecting groups; Azhayev et al, Tetrahedron 57, 4977-4986 (2001) foruniversal supports; Kozlov et al., Nucleosides, Nucleotides, and NucleicAcids, 24 (5-7), 1037-1041 (2005) for improved synthesis of longeroligonucleotides through the use of large-pore CPG; and Damha et al.,NAR, 18, 3813-3821 (1990) for improved derivitization).

Regardless of the type of synthesis, the resulting oligonucleotides maythen form the smaller building blocks for longer oligonucleotides. Asreferenced earlier, the smaller oligonucleotides can be joined togetherusing protocols known in the art, such as polymerase chain assembly(PCA), ligase chain reaction (LCR), and thermodynamically balancedinside-out synthesis (TBIO) (see Czar et al. Trends in Biotechnology,27, 63-71 (2009)). In PCA oligonucleotides spanning the entire length ofthe desired longer product are annealed and extended in multiple cycles(typically about 55 cycles) to eventually achieve full-length product.LCR uses ligase enzyme to join two oligonucleotides that are bothannealed to a third oligonucleotide. TBIO synthesis starts at the centerof the desired product and is progressively extended in both directionsby using overlapping oligos that are homologous to the forward strand atthe 5′ end of the gene and against the reverse strand at the 3′ end ofthe gene.

One method of the present invention provides an alternative method ofsynthesis of the smaller oligonucleotides. In this method, top-strandPCR (TSP), a plurality of oligonucleotides span the entire length of adesired product and are partially complementary to the adjacentoligonucleotide(s) (see FIG. 1). Amplification is performed withuniversal forward and reverse primers, and through multiple cycles ofamplification a full-length desired product is formed. This product canthen undergo dilution, sequence screening, and further amplificationthat results in the desired gene block end product.

In one method of TSP, the set of smaller oligonucleotides (“genesub-blocks”) that will be combined to form the full-length desiredproduct are between 40-200 bases long. In a further embodiment theoligonucleotide is between 75-200 bases, and in a further embodiment100-190 bases. The gene sub-blocks overlap each other by at least 15-20bases. For practical purposes, the overlap region should be at a minimumlong enough to ensure specific annealing of gene sub-blocks and have ahigh enough melting temperature (T_(m)) to anneal at the reactiontemperature employed. The overlap can extend to the point where a givengene sub-block is completely overlapped by adjacent gene sub-blocks. Theamount of overlap does not seem to have any effect on the quality of thefinal product. The first and last oligonucleotide building block in theassembly should contain binding sites for forward and reverseamplification primers. In one embodiment, the overlap regions containthe same sequence of complementarity to allow for the use of universalprimers.

Applicants have discovered that although intuitively more cycles of TSP(e.g., 30 cycles) would produce a greater percent of full-lengthmolecules, surprisingly a greater percent of correct full-lengthmolecules (i.e., assembled DNA strand having the desired sequencewithout error) is produced using a low number of cycles (about 15cycles). After the initial TSP reaction of ˜15 cycles, the product canthen be diluted 10-fold to 1000-fold, wherein the product is amplifiedagain in 20-30 cycles of PCR to increase mass yield of the desiredproduct.

Methods of mitigating synthesis errors are known in the art, and theyoptionally could be incorporated into methods of the present invention.The error correction methods include, but are not limited to,circularization methods wherein the properly assembled oligonucleotidesare circularized while the other product remain linear and wasenzymatically degraded (see Bang and Church, Nat. Methods, 5, 37-39(2008)). The mismatches can be degraded using mismatch-cleavingendonucleases. Another error correction method utilizes MutS proteinthat binds to mismatches, thereby allowing the desired product to beseparated (see Carr, P. A. et al. Nucleic Acids Res. 32, e162 (2004)).Other mismatch nucleases include those in the CEL nuclease family (seeSurveyor® Nuclease, Transgenomics), or RES I (see Errase™ synthetic geneerror correction kit, Novici). When error correction is used, the amountof remaining product may be small, and therefore a round of rescue PCRcan be performed to amplify the desired product.

Whether the gene sub-blocks are combined through TSP or another form ofassembly, the full-length product is diluted, typically to 0.25 to 10copies per reaction well, and then amplified to result in a large amountof homogeneous, desired double-stranded product (“gene block”). In afurther embodiment, the synthesized oligonucleotides are diluted to 1-5copies of the synthesized oligonucleotide per reaction well, and in afurther embodiment the synthesized oligonucleotides are diluted to 2-4copies per reaction well.

The dilution and amplification steps replace the conventional,time-consuming, labor-intensive in vivo cloning procedures traditionallyemployed, which are well known in the art. Some dilution methods areknown in the art (see Yehezkel et al., Nucleic Acids Research, vol. 36,no. 17, e107 (2008)), but they have been inefficient. The goal ofdilution PCR is to dilute the initial product, which is a mixture ofdesired perfect product and undesired imperfect products, into reactionwells to a dilution that best assures that an adequate amount ofreaction wells contain the desired product. If the mixture is too dilutethen there are undesired empty wells, and if the mixture is not dilutedenough then too many wells contain multiple template molecules (productof desired sequence plus defective product containing an undesiredsequence). Since DNA sequencing is a significant portion of genesynthesis cost, typical dilutions would normally be less than onemolecule per well, and even as low as 0.2 molecules per well to ensureclonality.

In the methods of the present invention, dilution conditions can be usedthat allow for as much as 1-5 copies of oligonucleotides/well to bepresent. Because the fidelity of the gene sub-blocks made using themethods of the present invention is so high, the product being dilutedis weighted heavily to contain a high percentage of correct sequencematerial. Therefore, if a given well has 4 molecules, there is still ahigh likelihood that the amplified product will be of the correctsequence. Diluting to an average copy of 1-5, or more preferably 2-4,reduces the number of empty wells and increases the efficiency of themanufacturing process.

The resulting product after dilution and amplification is then directlysequence verified without the traditional need to first clone theproduct into a plasmid vector and expand in a bacterial host. The finalproduct is a desired, sequence verified gene block. The gene blocks canthen later be cloned through methods well-known in the art, such asisothermal assembly (e.g., Gibson et al. Science, 319, 1215-1220(2008)); ligation-by-assembly or restriction cloning (e.g., Kodumal etal., Proc. Natl. Acad. Sci. U.S.A., 101, 15573-15578 (2004) andViallalobos et al., BMC Bioinformatics, 7, 285 (2006)); TOPO TA cloning(Invitrogen/Life Tech.); blunt-end cloning; and homologous recombination(e.g., Larionov et al., Proc. Natl. Acad. Sci. U.S.A., 93, 491-496). Thegene blocks can be cloned into many vectors known in the art, includingbut not limited to pUC57, pBluescriptII (Stratagene), pET27, Zero BluntTOPO (Invitrogen), psiCHECK-2, pIDTSMART (Integrated DNA Technologies,Inc.), and pGEM T (Promega).

The above methods can be re-ordered or altered, or further steps can beincorporated to optimize the end product, particularly if the knownend-product is shorter or longer. For example, in one embodiment wherethe desired end product is a gene block smaller than 500 bases, theinitial steps would be to synthesize the gene sub-blocks, perform PCR(e.g., TSP), then dilute and amplify. The resulting product is thentreated with error correction and then undergoes amplification, such asPCR. Optionally, the product undergoes a second dilution step andamplification. The end product then is re-amplified withsequence-specific primers if there is a need to remove universalsequences inserted for use in earlier amplification steps.

For longer desired products (i.e., greater than 500 bases), a number ofoptions are available to manufacture the longer end product. In oneembodiment, longer TSP-assembled starting material is used. In anotherembodiment, two or more smaller products are used, and those productsundergo isothermal assembly. Those products could be combined with otherproducts to make even longer gene blocks. In another embodiment, two ormore TSP-assembled products that undergo isothermal assembly. In anotherembodiment, a set of oligonucleotides of about 60 bases in length,wherein the oligonucleotides overlap with adjacent oligonucleotides tocover a 1-2 kb sequence length, are combined and undergo isothermalassembly.

The gene blocks can be used in a variety of applications, not limited tobut including protein expression (recombinant antibodies, novel fusionproteins, codon optimized short proteins, functional peptides—catalytic,regulatory, binding domains), microRNA genes, template for in vitrotranscription (IVT), shRNA expression cassettes, regulatory sequencecassettes, micro-array ready cDNA, gene variants and SNPs, DNA vaccines,standards for quantitative PCR and other assays, and functional genomics(mutant libraries and unrestricted point mutations for proteinmutagenesis, and deletion mutants). The ease of synthesizing large genesor gene segments using gene blocks allows for the synthesis of a set oflarge segments/genes wherein one or more gene blocks remain constantwhile one or more gene blocks varies.

The following examples further illustrate the invention but, of course,should not be construed as in any way limiting its scope.

Example 1

This example illustrates an assembly of gene sub-blocks into a productcontaining desired gene blocks.

A 935 bp gene block comprising 6 synthetic oligonucleotide subunits anda 1155 bp gene block comprising 8 synthetic oligonucleotide subunitswere assembled. The 8-oligonucleotide gene block is an extension of thesmaller 935 bp 6-oligonucleotide gene block sequence having anadditional 220 bp at the 3′-end. Therefore both gene blocks use the same6 component oligonucleotides in assembly, and the 8-oligonucleotide geneblock also includes oligonucleotides 7 and 8. The sequences of the geneblocks, the component oligonucleotides, the universal forward primer,the universal reverse primer (8-block) and gene specific reverse primers(6-block). Gene blocks were assembled using component oligonucleotideshaving an unmodified 3′-end, having 6 additional non-templated T basesadded to the 3′-end of each oligonucleotide (T-blocked), having 8additional non-templated T bases plus a terminal C3 spacer (propanediol)(T+C3-blocked), or having a terminal C3 spacer (propanediol) added tothe end of each oligonucleotide. The different end-blocked versions ofassembly will test whether extension from the 3′-end of componentoligonucleotides is necessary during TSP. The following sequences wereused:

935 base Gene Block (SEQ ID No. 1): CGACACTGCTCGATCCGCTCGCACCAAATCCAGATGACACAGAGTCCGTCCTCGCTTTCTGCGTCCCTGGGCGATCGTGTAACCATTACATGTCAGGCTAGTCGCGGCATCGGAAAAGACTTAAATTGGTACCAGCAGAAAGCGGGCAAAGCCCCTAAACTGCTGGTGAGCGATGCCAGCACATTGGAGGGCGGCGTTCCGTCACGTTTCAGTGGTAGCGGCTTCCATCAAAATTTCAGCTTAACCATCTCCAGTCTGCAGGCCGAGGATGTGGCTACCTATTTCTGCCAGCAGTATGAAACTTTCGGCCAGGGAACCAAAGTCGATATTAAAAGGTCGACGGTCGCGCCGAGCGTGTTTATTTTCCCGCCGTCTGATGAACAGCTGAAATCAGGCACCGCATCGGTGGTTTGCCTGCTGAACAATTTTTATCCGCGTGAAGCGAAAGTTCAGTGGAAAGTGGATAACGCCCTGCAGAGCGGTAATTCGCAAGAAAGCGTCACCGAACAAGATTCTAAAGACAGTACGTACTCCCTGAGCTCTACCCTGACGCTGTCAAAAGCAGATTACGAAAAACATAAAGTGTACGCTTGCGAAGTTACCCACCAAGGCCTGAGTTCCCCGGTTACGAAATCCTTCAACCGTGGCGAATGTTAAGCTGGGGATCCTCTAGAGGTTGAGGTGATTTTATGAAGAAAAACATTGCGTTTCTGCTGGCGAGCATGTTTGTGTTCTCTATCGCCACCAATGCGTATGCCCTCGAGCAAGTGCAATTGGTCCAGTCGGGCGCGGAAGTTAAGAAACCGGGGGCCTCTGTGAAAGTCTCCTGCAAAGCCTCTGGTTATACGTTTACAGGCTACTATATGCACTGGGTGCGTCAAGCCCCGGGTCAAGGTCTGGAGTGGATGGGTTGGATTAACCCGAACTCCGGTGGT 1155 base Gene Block (SEQ ID No. 2): CGACACTGCTCGATCCGCTCGCACCAAATCCAGATGACACAGAGTCCGTCCTCGCTTTCTGCGTCCCTGGGCGATCGTGTAACCATTACATGTCAGGCTAGTCGCGGCATCGGAAAAGACTTAAATTGGTACCAGCAGAAAGCGGGCAAAGCCCCTAAACTGCTGGTGAGCGATGCCAGCACATTGGAGGGCGGCGTTCCGTCACGTTTCAGTGGTAGCGGCTTCCATCAAAATTTCAGCTTAACCATCTCCAGTCTGCAGGCCGAGGATGTGGCTACCTATTTCTGCCAGCAGTATGAAACTTTCGGCCAGGGAACCAAAGTCGATATTAAAAGGTCGACGGTCGCGCCGAGCGTGTTTATTTTCCCGCCGTCTGATGAACAGCTGAAATCAGGCACCGCATCGGTGGTTTGCCTGCTGAACAATTTTTATCCGCGTGAAGCGAAAGTTCAGTGGAAAGTGGATAACGCCCTGCAGAGCGGTAATTCGCAAGAAAGCGTCACCGAACAAGATTCTAAAGACAGTACGTACTCCCTGAGCTCTACCCTGACGCTGTCAAAAGCAGATTACGAAAAACATAAAGTGTACGCTTGCGAAGTTACCCACCAAGGCCTGAGTTCCCCGGTTACGAAATCCTTCAACCGTGGCGAATGTTAAGCTGGGGATCCTCTAGAGGTTGAGGTGATTTTATGAAGAAAAACATTGCGTTTCTGCTGGCGAGCATGTTTGTGTTCTCTATCGCCACCAATGCGTATGCCCTCGAGCAAGTGCAATTGGTCCAGTCGGGCGCGGAAGTTAAGAAACCGGGGGCCTCTGTGAAAGTCTCCTGCAAAGCCTCTGGTTATACGTTTACAGGCTACTATATGCACTGGGTGCGTCAAGCCCCGGGTCAAGGTCTGGAGTGGATGGGTTGGATTAACCCGAACTCCGGTGGTACCAACTATGCGCAGAAATTCCAGGGTCGCGTCACGATGACTCGCGACACGTCAATTAGTACCGCGTACATGGAGTTATCGCGTTTACGTAGTGACGACACCGCCGTATACTACTGTGCGCGTGCTCAGAAACGCGGCCGTTCTGAATGGGCGTACGCACATTGGGGTCAAGGCACCCTGGTGACCGTGAGTAGTGGATCGACGAGAGCAGCGCG ACTGG Component oligonucleotide 1 (SEQ ID No. 3): CGACACTGCTCGATCCGCTCGCACCAAATCCAGATGACACAGAGTCCGTCCTCGCTTTCTGCGTCCCTGGGCGATCGTGTAACCATTACATGTCAGGCTAGTCGCGGCATCGGAAAAGACTTAAATTGGTACCAGCAGAAAGCGGGCAAAGCCCCTAAACTGCTGGTGAGCGATGCCAGCACATTGGAG Component oligonucleotide 2 (SEQ ID No. 4): CTGGTGAGCGATGCCAGCACATTGGAGGGCGGCGTTCCGTCACGTTTCAGTGGTAGCGGCTTCCATCAAAATTTCAGCTTAACCATCTCCAGTCTGCAGGCCGAGGATGTGGCTACCTATTTCTGCCAGCAGTATGAAACTTTCGGCCAG GGAACCAAAGTC Component oligonucleotide 3 (SEQ ID No. 5): GCAGTATGAAACTTTCGGCCAGGGAACCAAAGTCGATATTAAAAGGTCGACGGTCGCGCCGAGCGTGTTTATTTTCCCGCCGTCTGATGAACAGCTGAAATCAGGCACCGCATCGGTGGTTTGCCTGCTGAACAATTTTTATCCGCGTGAAGCGAAAGTTCAGTGGAAAGTGGATAACGCCCTGCAGAG Component oligonucleotide 4 (SEQ ID No. 6): GTTCAGTGGAAAGTGGATAACGCCCTGCAGAGCGGTAATTCGCAAGAAAGCGTCACCGAACAAGATTCTAAAGACAGTACGTACTCCCTGAGCTCTACCCTGACGCTGTCAAAAGCAGATTACGAAAAACATAAAGTGTACGCTTGCGAAGTTACCCACCAAGGCCTGAGTTCCCCGGTTACGAAATCC Component oligonucleotide 5 (SEQ ID No. 7): CCAAGGCCTGAGTTCCCCGGTTACGAAATCCTTCAACCGTGGCGAATGTTAAGCTGGGGATCCTCTAGAGGTTGAGGTGATTTTATGAAGAAAAACATTGCGTTTCTGCTGGCGAGCATGTTTGTGTTCTCTATCGCCACCAATGCGTATGCCCTCGAGCAAGTGCAATTGGTC  Component oligonucleotide 6 (SEQ ID No. 8): CGTATGCCCTCGAGCAAGTGCAATTGGTCCAGTCGGGCGCGGAAGTTAAGAAACCGGGGGCCTCTGTGAAAGTCTCCTGCAAAGCCTCTGGTTATACGTTTACAGGCTACTATATGCACTGGGTGCGTCAAGCCCCGGGTCAAGGTCTGGAGTGGATGGGTTGGATTAACCCGAACTCCGGTGGT Component oligonucleotide 7 (SEQ ID No. 9): GGGTTGGATTAACCCGAACTCCGGTGGTACCAACTATGCGCAGAAATTCCAGGGTCGCGTCACGATGACTCGCGACACGTCAATTAGTACCGCGTACATGGAGTTATCGCGTTTACGTAGTG  Component oligonucleotide 8 (SEQ ID No. 10): TACCGCGTACATGGAGTTATCGCGTTTACGTAGTGACGACACCGCCGTATACTACTGTGCGCGTGCTCAGAAACGCGGCCGTTCTGAATGGGCGTACGCACATTGGGGTCAAGGCACCCTGGTGACCGTGAGTAGTGGATCGACGAGAGC AGCGCGACTGG Universal For primer 5′-phos (SEQ ID No. 11):/5Phos/CGACACTGCTCGATCCGCTCGCACC Universal Rev primer 5′-phos (SEQ ID No. 12):/5Phos/CCAGTCGCGCTGCTCTCGTCGATCC Gene Specific Rev primer (SEQ ID No. 13):/5Phos/ACCACCGGAGTTCGGGTTAATCCAACC 

The component oligonucleotides were assembled by TSP using the followingreaction mixture and conditions:

50/100 nM component oligonucleotides 1-6/850/100 nM forward primer200 nM reverse primer0.02 U/uL KOD Hot-Start DNA polymerase (Novagen)1× buffer for KOD Hot Start DNA polymerase (Novagen)

1.5 mM MgSO₄

0.8 mM dNTPs (0.2 mM each)Cycling conditions: 95° C.^(3:00) (95° C.^(0:20)−70° C.^(0:30))×15, 20,25, or 30 cycles

Additionally, several sets of otherwise identical componentoligonucleotides were used in TSP assembly wherein the 3′ ends areeither unblocked or blocked with a 6-residue poly-T; a C3 spacer; or a8-residue poly-T plus a C3 spacer. After the TSP cycles, the resultingproducts were diluted 100-fold in water, and then underwent a furtherstep of PCR containing 200 nM each of the universal forward primer and200 nM of the universal reverse primer (8-block) or gene specificreverse primer (6-block) (cycling conditions: 95° C.^(3:00) (95°C.^(0:20)−70° C.^(0:30))×30 cycles). The resulting gene block was run ona 1.2% agarose gel at 100V for 1 hour 30 minutes.

FIG. 2A is a picture of the resulting gel of the 935-base 6-componentoligonucleotides gene block, and FIG. 2B is the resulting gel for the1155 base 8-component oligonucleotides gene block. Notably, at 15 TSPcycles the desired product is present, particularly using the unblockedcomponent oligonucleotides. The 50 nM 6-block gene blocks were bluntcloned into a pUC19 vector and sequence verified using Sanger sequencingusing a 3730xl sequencer (Life Technologies-Applied Biosystems). FIG. 3Agraphically represents the percent of full length clones with no errorsfor each set of reactions. FIG. 3B represents the percent of all cloneswith no errors after sequencing additional clones generated usingunblocked component oligonucleotides. Although all conditions producefull-length clones with no errors, surprisingly the low cycle numberunblocked component oligonucleotides was the most robust and yielded thehighest percentage of correct final products.

Example 2

This example demonstrates that various lengths, concentrations andnumbers of component oligonucleotides and varying componentoligonucleotide overlap conditions can be used to successfully producefull length gene blocks.

FIG. 4 illustrates the three overlap designs used in this example.Design 1 uses component oligonucleotides that are 115-185 nucleotides inlength. Design 2 uses shorter component oligonucleotides, between 54-125nucleotides. Design 3 uses 100 nucleotide-long componentoligonucleotides (the last oligonucleotide may be shorter or longer than100 nucleotides depending on final gene block length), and employs acomplete overlap design wherein the adjacent oligonucleotides completelyoverlap each non-terminal component.

The following are the sequences of the desired gene blocks and componentoligonucleotides. The same universal For and Rev primers from Example 1(SEQ ID Nos. 11-12) were used.

Gene Block A (SEQ ID No. 14): CGACACTGCTCGATCCGCTCGCACCTTTCTGGCATGAGGTCACTGACAGCCCTCTGGACAACACAGCTTATTTATTGGTCTCTCATTCTCCCATCCCCACTCCTCCTTTCTTCCCTCTCTCCACCAGAGCGATGGCGTCACCGGCCCATCCTCCAAGCCGGACTGCCGGCAAATGCCTCCACAGTGGTCGGAGGAGACGTAGAGTTTGTCTGCAAGGTTTACAGTGATGCCCAGCCCCACATCCAGTGGATCAAGCACGTGGAAAAGAACGGCAGTAAATACGGGCCCGACGGGCTGCCCTACCTCAAGGTTCTCAAGGTGAGGACTTTCTGAATCTAAAGGTACCCACAACTGGGGTCTCCTTCATGGGTTTGGCCACAGGTTCTTTGATTTCCTGTTGGAGTTGAGAGAGGATGATTCTCTTTTTTGACTAGCCAGCAGAGAGTGTTCTAAGGGGATCGACGAGAGCAGCGCGACTGG  Gene Block B (SEQ ID No. 15): CGACACTGCTCGATCCGCTCGCACCGCACCTGTACGATCACTGAACTGCAGAATCTGGGATGTTAACCAGAAGACCTTCTATCTGAGGAACAACCAACTAGTTGCTGGATACTTGCAAGGACCAAATGTCAATTTAGAAGAAAAGTTCGACATGTCCTTTGTAGAAGGACATGAAAGTAATGACAAAATACCTGTGGCCTTGGGCCTCAAGGAAAAGAATCTGTACCTGTCCTGCGTGTTGAAAGATGATGAACCCACTCTACAGCTGGAGGCTGTAAATCCCAAAAATTACCCAAAGAGGAAGATGGAAAAGCGATTTGTCTTCAACAAGATAGATTCAGGCCCAACCACATCATTTGAGTCTGCCCAGTTCCCCAACTGGTTCCTCTGCACAGCGATGGAAGCTGACCAGCCCGTCAGCCTCACCAATATGCCTGACGAAGGCGTCATGGTCACCAAATTCTACATGCAATTTGTGTCTTCCGGATCGACGAGAGCAG CGCGACTGG Gene Block C (SEQ ID No. 16): CGACACTGCTCGATCCGCTCGCACCCATATGGCCAAGCTGACCAGCGCCGTTCCGGTGCTCACCGCGCGCGACGTCGCCGGAGCGGTCGAGTTCTGGACCGACCGGCTCGGGTTCTCCCGGGACTTCGTGGAGGACGACTTCGCCGGTGTGGTCCGGGACGACGTGACCCTGTTCATCAGCGCGGTCCAGGACCAGGTAAGACGCAATTCTGCTGTGCACGTGCCAATGCCGCTGCCCCCCAGCGCATTGGCTCACCATCGCCATCGCCATTGCTGCTGCAGGTGGTGCCGGACAACACCCTGGCCTGGGTGTGGGTGCGCGGCCTGGACGAGCTGTACGCCGAGTGGTCGGAGGTCGTGTCCACGAACTTCCGGGACGCCTCCGGGCCGGCCATGACCGAGATCGGCGAGCAGCCGTGGGGGCGGGAGTTCGCCCTGCGCGACCCGGCCGGCAACTGCGTGCACTTCGTGGCCGAGGAGCAGGACTAAGGATCCGGATC GACGAGAGCAGCGCGACTGG Gene Block A Design 1 Component oligonucleotide 1 (SEQ ID No. 17): CGACACTGCTCGATCCGCTCGCACCTTTCTGGCATGAGGTCACTGACAGCCCTCTGGACAACACAGCTTATTTATTGGTCTCTCATTCTCCCATCCCCACTCCTCCTTTCTTCCCTCTCTCCACCAGAGCGATGGCGTCACCGGCCCATCCTCCAAGCCGGACTGCCGGCAAATGCCTCCACAG Gene Block A Design 1 Component oligonucleotide 2 (SEQ ID No. 18): GGACTGCCGGCAAATGCCTCCACAGTGGTCGGAGGAGACGTAGAGTTTGTCTGCAAGGTTTACAGTGATGCCCAGCCCCACATCCAGTGGATCAAGCACGTGGAAAAGAACGGCAGTAAATACGGGCCCGACGGGCTGCCCTACCTCAAGGTTCTCAAGGTGAGGACTTTCTGAATCTAAAGG Gene Block A Design 1 Component oligonucleotide 3 (SEQ ID No. 19): CCTCAAGGTTCTCAAGGTGAGGACTTTCTGAATCTAAAGGTACCCACAACTGGGGTCTCCTTCATGGGTTTGGCCACAGGTTCTTTGATTTCCTGTTGGAGTTGAGAGAGGATGATTCTCTTTTTTGACTAGCCAGCAGAGAGTGTTCTAAGGGGATCGACGAGAGCAGCGCGACTGG Gene Block A Design 2 Component oligonucleotide 1 (SEQ ID No. 20): CGACACTGCTCGATCCGCTCGCACCTTTCTGGCATGAGGTCACTGACAGC CCTCTGG Gene Block A Design 2 Component oligonucleotide 2 (SEQ ID No. 21): GGCATGAGGTCACTGACAGCCCTCTGGACAACACAGCTTATTTATTGGTCTCTCATTCTCCCATCCCCACTCCTCCTTTCTTCCCTCTCTCCACCAGAGCGATGGCGTCACCGGCCCATCC  Gene Block A Design 2 Component oligonucleotide 3(SEQ ID No. 22):  CGATGGCGTCACCGGCCCATCCTCCAAGCCGGACTGCCGGCAAATGCCTCCACAGTGGTCGGAGGAGACGTAGAGTTTGTCTGCAAGGTTTACAGTGATG Gene Block A Design 2 Component oligonucleotide 4 (SEQ ID No. 23): GGAGACGTAGAGTTTGTCTGCAAGGTTTACAGTGATGCCCAGCCCCACATCCAGTGGATCAAGCACGTGGAAAAGAACGGCAGTAAATACGGGCCCGACG GGCTGCCCTACC Gene Block A Design 2 Component oligonucleotide 5 (SEQ ID No. 24): GCCCGACGGGCTGCCCTACCTCAAGGTTCTCAAGGTGAGGACTTTCTGAATCTAAAGGTACCCACAACTGGGGTCTCCTTCATGGGTTTGGCCACAGGTT CTTTGATTTCCTGTTGGAG Gene Block A Design 2 Component oligonucleotide 6 (SEQ ID No. 25): GGTTTGGCCACAGGTTCTTTGATTTCCTGTTGGAGTTGAGAGAGGATGATTCTCTTTTTTGACTAGCCAGCAGAGAGTGTTCTAAGGGGATCGACGAGAG CAGCGCGACTGG Gene Block A Design 3 Component oligonucleotide 1 (SEQ ID No. 26): CGACACTGCTCGATCCGCTCGCACCTTTCTGGCATGAGGTCACTGACAGCCCTCTGGACAACACAGCTTATTTATTGGTCTCTCATTCTCCCATCCCCAC Gene Block A Design 3 Component oligonucleotide 2 (SEQ ID No. 27): CCTCTGGACAACACAGCTTATTTATTGGTCTCTCATTCTCCCATCCCCACTCCTCCTTTCTTCCCTCTCTCCACCAGAGCGATGGCGTCACCGGCCCATC Gene Block A Design 3 Component oligonucleotide 3 (SEQ ID No. 28): TCCTCCTTTCTTCCCTCTCTCCACCAGAGCGATGGCGTCACCGGCCCATCCTCCAAGCCGGACTGCCGGCAAATGCCTCCACAGTGGTCGGAGGAGACGT Gene Block A Design 3 Component oligonucleotide 4 (SEQ ID No. 29): CTCCAAGCCGGACTGCCGGCAAATGCCTCCACAGTGGTCGGAGGAGACGTAGAGTTTGTCTGCAAGGTTTACAGTGATGCCCAGCCCCACATCCAGTGGA Gene Block A Design 3 Component oligonucleotide 5 (SEQ ID No. 30): AGAGTTTGTCTGCAAGGTTTACAGTGATGCCCAGCCCCACATCCAGTGGATCAAGCACGTGGAAAAGAACGGCAGTAAATACGGGCCCGACGGGCTGCCC Gene Block A Design 3 Component oligonucleotide 6 (SEQ ID No. 31): TCAAGCACGTGGAAAAGAACGGCAGTAAATACGGGCCCGACGGGCTGCCCTACCTCAAGGTTCTCAAGGTGAGGACTTTCTGAATCTAAAGGTACCCACA Gene Block A Design 3 Component oligonucleotide 7 (SEQ ID No. 32): TACCTCAAGGTTCTCAAGGTGAGGACTTTCTGAATCTAAAGGTACCCACAACTGGGGTCTCCTTCATGGGTTTGGCCACAGGTTCTTTGATTTCCTGTTG Gene Block A Design 3 Component oligonucleotide 8 (SEQ ID No. 33): ACTGGGGTCTCCTTCATGGGTTTGGCCACAGGTTCTTTGATTTCCTGTTGGAGTTGAGAGAGGATGATTCTCTTTTTTGACTAGCCAGCAGAGAGTGTTC Gene Block A Design 3 Component oligonucleotide 9 (SEQ ID No. 34): GAGTTGAGAGAGGATGATTCTCTTTTTTGACTAGCCAGCAGAGAGTGTTCTAAGGGGATCGACGAGAGCAGCGCGACTGG Gene Block B Design 1 Component oligonucleotide 1 (SEQ ID No. 35): CGACACTGCTCGATCCGCTCGCACCGCACCTGTACGATCACTGAACTGCAGAATCTGGGATGTTAACCAGAAGACCTTCTATCTGAGGAACAACCAACTAGTTGCTGGATACTTGCAAGGACCAAATGTCAATTTAGAAG Gene Block B Design 1 Component oligonucleotide 2 (SEQ ID No. 36): GTTGCTGGATACTTGCAAGGACCAAATGTCAATTTAGAAGAAAAGTTCGACATGTCCTTTGTAGAAGGACATGAAAGTAATGACAAAATACCTGTGGCCTTGGGCCTCAAGGAAAAGAATCTGTACCTGTCCTGCGTGTTGAAAGATGATGAACCCACTCTACAGCTGGAGGCTGTAAATCCC Gene Block B Design 1 Component oligonucleotide 3 (SEQ ID No. 37): GAACCCACTCTACAGCTGGAGGCTGTAAATCCCAAAAATTACCCAAAGAGGAAGATGGAAAAGCGATTTGTCTTCAACAAGATAGATTCAGGCCCAACCACATCATTTGAGTCTGCCCAGTTCCCCAACTGGTTCCTCTGCACAGCGATG GAAGCTG Gene Block B Design 1 Component oligonucleotide 4 (SEQ ID No. 38): CTGGTTCCTCTGCACAGCGATGGAAGCTGACCAGCCCGTCAGCCTCACCAATATGCCTGACGAAGGCGTCATGGTCACCAAATTCTACATGCAATTTGTGTCTTCCGGATCGACGAGAGCAGCGCGACTGG Gene Block B Design 2 Component oligonucleotide 1 (SEQ ID No. 39): CGACACTGCTCGATCCGCTCGCACCGCACCTGTACGATCACTGAACTGCAGAATCTGGGATGTTAACCAGAAG Gene Block B Design 2 Component oligonucleotide 2 (SEQ ID No. 40): CGATCACTGAACTGCAGAATCTGGGATGTTAACCAGAAGACCTTCTATCTGAGGAACAACCAACTAGTTGCTGGATACTTGCAAGGACCAAATGTCAATTTAGAAGAAAAGTTCGACATGTCC Gene Block B Design 2 Component oligonucleotide 3 (SEQ ID No. 41): ATGTCAATTTAGAAGAAAAGTTCGACATGTCCTTTGTAGAAGGACATGAAAGTAATGACAAAATACCTGTGGCCTTGGGCCTCAAGG Gene Block B Design 2 Component oligonucleotide 4 (SEQ ID No. 42): ACCTGTGGCCTTGGGCCTCAAGGAAAAGAATCTGTACCTGTCCTGCGTGTTGAAAGATGATGAACCCACTCTACAGCTGGAGGCTGTAAATCCC Gene Block B Design 2 Component oligonucleotide 5 (SEQ ID No. 43): GAACCCACTCTACAGCTGGAGGCTGTAAATCCCAAAAATTACCCAAAGAGGAAGATGGAAAAGCGATTTGTCTTCAACAAGATAGATTCAGGCCCAACCACATCATTTGAGTCTGCCCAGTTCCC Gene Block B Design 2 Component oligonucleotide 6 (SEQ ID No. 44): AACCACATCATTTGAGTCTGCCCAGTTCCCCAACTGGTTCCTCTGCACAGCGATGGAAGCTGACCAGCCCGTCAGCCTCACCAATATGCCTGACGAAGGC Gene Block B Design 2 Component oligonucleotide 7 (SEQ ID No. 45): GTCAGCCTCACCAATATGCCTGACGAAGGCGTCATGGTCACCAAATTCTACATGCAATTTGTGTCTTCCGGATCGACGAGAGCAGCGCGACTGG Gene Block B Design 3 Component oligonucleotide 1 (SEQ ID No. 46): CGACACTGCTCGATCCGCTCGCACCGCACCTGTACGATCACTGAACTGCAGAATCTGGGATGTTAACCAGAAGACCTTCTATCTGAGGAACAACCAACTA Gene Block B Design 3 Component oligonucleotide 2 (SEQ ID No. 47): GAATCTGGGATGTTAACCAGAAGACCTTCTATCTGAGGAACAACCAACTAGTTGCTGGATACTTGCAAGGACCAAATGTCAATTTAGAAGAAAAGTTCGA Gene Block B Design 3 Component oligonucleotide 3 (SEQ ID No. 48): AGTTGCTGGATACTTGCAAGGACCAAATGTCAATTTAGAAGAAAAGTTCGACATGTCCTTTGTAGAAGGACATGAAAGTAATGACAAAATACCTGTGGCC Gene Block B Design 3 Component oligonucleotide  (SEQ ID No. 49): ACATGTCCTTTGTAGAAGGACATGAAAGTAATGACAAAATACCTGTGGCCTTGGGCCTCAAGGAAAAGAATCTGTACCTGTCCTGCGTGTTGAAAGATGA Gene Block B Design 3 Component oligonucleotide 5 (SEQ ID No. 50): CTTGGGCCTCAAGGAAAAGAATCTGTACCTGTCCTGCGTGTTGAAAGATGATGAACCCACTCTACAGCTGGAGGCTGTAAATCCCAAAAATTACCCAAAG Gene Block B Design 3 Component oligonucleotide 6 (SEQ ID No. 51): ATGAACCCACTCTACAGCTGGAGGCTGTAAATCCCAAAAATTACCCAAAGAGGAAGATGGAAAAGCGATTTGTCTTCAACAAGATAGATTCAGGCCCAAC Gene Block B Design 3 Component oligonucleotide 7 (SEQ ID No. 52): GAGGAAGATGGAAAAGCGATTTGTCTTCAACAAGATAGATTCAGGCCCAACCACATCATTTGAGTCTGCCCAGTTCCCCAACTGGTTCCTCTGCACAGCG Gene Block B Design 3 Component oligonucleotide 8 (SEQ ID No. 53): CCACATCATTTGAGTCTGCCCAGTTCCCCAACTGGTTCCTCTGCACAGCGATGGAAGCTGACCAGCCCGTCAGCCTCACCAATATGCCTGACGAAGGCGT Gene Block B Design 3 Component oligonucleotide 9 (SEQ ID No. 54): GATGGAAGCTGACCAGCCCGTCAGCCTCACCAATATGCCTGACGAAGGCGTCATGGTCACCAAATTCTACATGCAATTTGTGTCTTCCGGATCGACGAGA GCAGCGCGACTGG Gene Block C Design 1 Component oligonucleotide 1 (SEQ ID No. 55): CGACACTGCTCGATCCGCTCGCACCCATATGGCCAAGCTGACCAGCGCCGTTCCGGTGCTCACCGCGCGCGACGTCGCCGGAGCGGTCGAGTTCTGGACCGACCGGCTCGGGTTCTCCCGGGACTTCGTGGAGGA Gene Block C Design 1 Component oligonucleotide 2 (SEQ ID No. 56): GGGTTCTCCCGGGACTTCGTGGAGGACGACTTCGCCGGTGTGGTCCGGGACGACGTGACCCTGTTCATCAGCGCGGTCCAGGACCAGGTAAGACGCAATTCTGCTGTGCACGTGCCAATGCCGCTGCCCCCCAGCGCATTGGCTCACCAT CGCCATCGCCATTG Gene Block C Design 1 Component oligonucleotide 3 (SEQ ID No. 57): CATTGGCTCACCATCGCCATCGCCATTGCTGCTGCAGGTGGTGCCGGACAACACCCTGGCCTGGGTGTGGGTGCGCGGCCTGGACGAGCTGTACGCCGAG TGGTCGGAGGTCGTG Gene Block C Design 1 Component oligonucleotide 4 (SEQ ID No. 58): GTACGCCGAGTGGTCGGAGGTCGTGTCCACGAACTTCCGGGACGCCTCCGGGCCGGCCATGACCGAGATCGGCGAGCAGCCGTGGGGGCGGGAGTTCGCCCTGCGCGACCCGGCCGGCAACTGCGTGCACTTCGTGGCCGAGGAGCAGGACTAAGGATCCGGATCGACGAGAGCAGCGCGACTGG Gene Block C Design 2 Component oligonucleotide 1 (SEQ ID No. 59): CGACACTGCTCGATCCGCTCGCACCCATATGGCCAAGCTGACCAGCGCCG TTCC Gene Block C Design 2 Component oligonucleotide 2 (SEQ ID No. 60): GCCAAGCTGACCAGCGCCGTTCCGGTGCTCACCGCGCGCGACGTCGCCGGAGCGGTCGAGTTCTGGACCGACCGGCTCGGGTTCTCCCGGGACTTCGTGGAGGACGACTTCGCCGGTGTGG  Gene Block C Design 2 Component oligonucleotide 3(SEQ ID No. 61):  TGGAGGACGACTTCGCCGGTGTGGTCCGGGACGACGTGACCCTGTTCATCAGCGCGGTCCAGGACCAGGTAAGACGCAATTCTGCTG Gene Block C Design 2 Component oligonucleotide 4 (SEQ ID No. 62): GTCCAGGACCAGGTAAGACGCAATTCTGCTGTGCACGTGCCAATGCCGCTGCCCCCCAGCGCATTGGCTCACCATCGCCATCGCCATTGCTG Gene Block C Design 2 Component oligonucleotide 5 (SEQ ID No. 63): CACCATCGCCATCGCCATTGCTGCTGCAGGTGGTGCCGGACAACACCCTGGCCTGGGTGTGGGTGCGCGGCCTGGACGAGCTGTACGCCGAGTGGTCGGA GGTC Gene Block C Design 2 Component oligonucleotide 6 (SEQ ID No. 64): GCTGTACGCCGAGTGGTCGGAGGTCGTGTCCACGAACTTCCGGGACGCCTCCGGGCCGGCCATGACCGAGATCGGCGAGCAGCC Gene Block C Design 2 Component oligonucleotide 7 (SEQ ID No. 65): CCATGACCGAGATCGGCGAGCAGCCGTGGGGGCGGGAGTTCGCCCTGCGCGACCCGGCCGGCAACTGCGTGCACTTCGTGGCCGAGGAGCAGGACTAAGG Gene Block C Design 2 Component oligonucleotide 8 (SEQ ID No. 66): CACTTCGTGGCCGAGGAGCAGGACTAAGGATCCGGATCGACGAGAGCAGC GCGACTGG Gene Block C Design 3 Component oligonucleotide 1 (SEQ ID No. 67): CGACACTGCTCGATCCGCTCGCACCCATATGGCCAAGCTGACCAGCGCCGTTCCGGTGCTCACCGCGCGCGACGTCGCCGGAGCGGTCGAGTTCTGGACC Gene Block C Design 3 Component oligonucleotide 2 (SEQ ID No. 68): TTCCGGTGCTCACCGCGCGCGACGTCGCCGGAGCGGTCGAGTTCTGGACCGACCGGCTCGGGTTCTCCCGGGACTTCGTGGAGGACGACTTCGCCGGTGT Gene Block C Design 3 Component oligonucleotide 3 (SEQ ID No. 69): CGACCGGCTCGGGTTCTCCCGGGACTTCGTGGAGGACGACTTCGCCGGTGTGGTCCGGGACGACGTGACCCTGTTCATCAGCGCGGTCCAGGACCAGGTA Gene Block C Design 3 Component oligonucleotide 4 (SEQ ID No. 70): TGGTCCGGGACGACGTGACCCTGTTCATCAGCGCGGTCCAGGACCAGGTAAGACGCAATTCTGCTGTGCACGTGCCAATGCCGCTGCCCCCCAGCGCATT Gene Block C Design 3 Component oligonucleotide 5 (SEQ ID No. 71): AAGACGCAATTCTGCTGTGCACGTGCCAATGCCGCTGCCCCCCAGCGCATTGGCTCACCATCGCCATCGCCATTGCTGCTGCAGGTGGTGCCGGACAACA Gene Block C Design 3 Component oligonucleotide 6 (SEQ ID No. 72): TGGCTCACCATCGCCATCGCCATTGCTGCTGCAGGTGGTGCCGGACAACACCCTGGCCTGGGTGTGGGTGCGCGGCCTGGACGAGCTGTACGCCGAGTGG Gene Block C Design 3 Component oligonucleotide 7 (SEQ ID No. 73): ACCCTGGCCTGGGTGTGGGTGCGCGGCCTGGACGAGCTGTACGCCGAGTGGTCGGAGGTCGTGTCCACGAACTTCCGGGACGCCTCCGGGCCGGCCATGA Gene Block C Design 3 Component oligonucleotide 8 (SEQ ID No. 74): GTCGGAGGTCGTGTCCACGAACTTCCGGGACGCCTCCGGGCCGGCCATGACCGAGATCGGCGAGCAGCCGTGGGGGCGGGAGTTCGCCCTGCGCGACCCG Gene Block C Design 3 Component oligonucleotide 9 (SEQ ID No. 75): ACCGAGATCGGCGAGCAGCCGTGGGGGCGGGAGTTCGCCCTGCGCGACCCGGCCGGCAACTGCGTGCACTTCGTGGCCGAGGAGCAGGACTAAGGATCCGGATCGACGAGAGCAGCGCGACTGG 

The Component oligonucleotides were pooled, assembled using TSP, dilutedand subsequently amplified with PCR as was done previously in Example 1.As in Example 1, the resulting product was run on gels (see FIG. 5) andDNA sequence of the amplification product was determined for gene blocksgenerated using 25 nM or 100 nM of each component oligonucleotide andrun for 15 TSP cycles (see FIGS. 6A and 6B). As the gels and graphsindicate, each design method, at varying cycles, produces the desiredgene block product. While any of the assembly methods outlined above canbe used with the method of the invention, use of the longer componentoligonucleotides with less overlap is preferred to simplify design andautomation as well as lowering materials cost. Thus fewer, longercomponent oligonucleotides can be utilized under short cyclingconditions to assemble the desired gene block.

Example 3

This example demonstrates the synthesis of a gene block using TSP,followed by dilution and subsequent amplification of the desiredsequence gene block from a heterogeneous assembly mixture.

Example 3 Gene Block (SEQ ID No. 76)

ACCGGTTCCTGGGCCCAGTCTGCCCTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCATCTCCTGCAATGGAACCAGCAGTGACGTTGGTGGATTTGACTCTGTCTCCTGGTACCAACAGCACCCAGGCAAAGCCCCCAAACTCATGATTTATGATGTCAGTCATCGGCCCTCAGGGGTTTCTAATCGCTTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCCTGACCATCTCTGGGCTCCAGGCTGAGGACGAGGCTGATTATTACTGCTCTTCACTGACAGACAGAAGCCATCGCATATTCGGCGGAGGGACCAAGCTGACCGTCCTAGGTCAGCCCAAGGCTGCCCCCTCGGTCACTCTGTTCCCGCCCTCGAG

Example 3 Component Oligonucleotides

SEQ ID No. 77: CGACACTGCTCGATCCGCTCGCACCACCGGTTCCTGGGCCCAGTCTGCCCTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCATCTCCTGCAATGGAACCAGCAGTGACGTTGGTGGATTTGACTCTGTCTCCTGGTACCAACAGCACCCAGGCAAAG SEQ ID No. 78:CTCCTGGTACCAACAGCACCCAGGCAAAGCCCCCAAACTCATGATTTATGATGTCAGTCATCGGCCCTCAGGGGTTTCTAATCGCTTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCCTGACCATCTCTGGGCTCCAGGCTGAGGA CGAG SEQ ID No. 79:CTCTGGGCTCCAGGCTGAGGACGAGGCTGATTATTACTGCTCTTCACTGACAGACAGAAGCCATCGCATATTCGGCGGAGGGACCAAGCTGACCGTCCTAGGTCAGCCCAAGGCTGCCCCCTCGGTCACTCTGTTCCCGCCCTCGAGGGATCGACGAGAGCAGCGCGACTGG Forward primer (SEQ ID No. 80):/5Phos/ACCGGTTCCTGGGCC (59.3° Tm) Reverse Primer (SEQ ID No. 81):/5Phos/CTCGAGGGCGGGAACAG (60.1° Tm)

There is a 29-base overlap between the first and second componentoligonucleotides (70.1°Tm), and a 25-base overlap between the second andthird component oligonucleotides (69.4°Tm). TSP assembly was performedon a disposable tip Janus robot, the product was then diluted (5 μl ofthe final product of the TSP cycling diluted into 145 μl of water), andthe diluted aliquot was further amplified by PCR using the indicatedterminal Forward and Reverse primers.

TSP Reaction Mixture

The TSP reaction was set up in a final reaction volume of 25 μL. Eacholigonucleotide (including the forward and reverse universal primers)were at a final concentration of 140 nM in a 1×KOD DNA polymerasebuffer. The reaction contained a final concentration of 0.8 mM dNTPs,1.5 mM MgSO₄ and 0.5 U of KOD DNA polymerase (Novagen). The cyclingparameters were: 95^(3:00)−(95^(0:15)−70^(0:30))×30. After the initialcycling, the reaction was diluted 1:5 and was reamplified with theaddition of fresh forward and reverse universal primers, MgSO₄, anddNTPs under the same cycling conditions.

The amplification products were separated by agarose gel electrophoresisand visualized by fluorescent dye staining to verify the length of theassembled gene block. Sample cleanup was performed (QIAquick PCRPurification Kit, Qiagen) and the product was quantified via UVabsorbance (Abs=0.034, Conc.=22.1 ng/uL). Serial dilution was performed(Janus DT robot) with IDTE buffer 8.0 pH w/tRNA at a conc. 0.1 mg/ml fora final dilution of 0.3 copies/5 μL.

Serial Dilutions:

1=5 uL of Sample, 145 μL of diluent, MIX

2=5 uL of first dilution, 71 μL of diluent, MIX

3=15 uL of 2^(nd) dilution, 145 μL of diluent, MIX

4=15 uL of 3^(rd) dilution, 145 μL of diluent, MIX

5=15 uL of 4^(th) dilution, 145 μL of diluent, MIX

6=15 uL of 5^(th) dilution, 145 μL of diluent, MIX

7=15 uL of 6^(th) dilution, 145 μL of diluent, MIX

8=15 uL of 7^(th) dilution, 145 μL of diluent, MIX

9=15 uL of 8^(th) dilution, 145 μL of diluent, MIX

10=15 uL of 9^(th) dilution, 145 μL of diluent, MIX

11=15 uL of 10^(th) dilution, 145 μL of diluent, MIX

Dilution PCR: Plates containing 1×KOD buffer, 0.25 U KOD DNA polymerase,1.5 mM MgSO₄, 300 nM forward and reverse universal primers, and 0.6×EvaGreen, 5 μL of the final dilution of the assembled gene block, all ina final volume of 25 μL. The cycling conditions were95^(2:00)−(95^(0:20)−70^(1:00))×45.

Wells containing a positive fluorescent signal were diluted 1:16 inwater. The diluted amplified product was then sequence verified usingstandard Sanger based sequencing on a 3730XL DNA sequencer. One μL ofthe diluted clonally amplified product was further amplified by PCRusing gene specific terminal primers under the following conditions;1×KOD buffer, 0.5 U KOD DNA polymerase, 1.5 mM MgSO₄, 0.8 mM dNTPs, and200 nM primers. The cycling conditions were95^(3:00)−(95^(0:15)−60^(0:15)−70^(0:30))×30 cycles.

The TSP generated gene block and dilution-amplified gene block wereseparated using agarose gel electrophoresis and visualized byfluorescent dye staining. A 1 kb marker size ladder (Axygen) wasincluded (see FIG. 7). The size markers are shown for the four smalleststandards. The expected product is 442 bp, and products of this sizewere visualized on the gel.

Example 4

The present example demonstrates an alternate protocol wherein the TSPcycling conditions are modified to allow for longer extension times tomake longer gene blocks. The longer cycling conditions significantlyimprove the resulting desired product.

The 8 sub-block gene block (1155 bases, SEQ ID No. 2) from Example 1 wassynthesized, as well as a 10 sub-block gene block (1308 bases, SEQ IDNo. 82). The 10 sub-blocks are SEQ ID Nos. 83-92.

The component oligonucleotides were assembled by TSP using the followingreaction mixture and conditions:

50 nM component oligonucleotides50 nM forward primer200 nM reverse primer0.02 U/uL KOD Hot-Start DNA polymerase (Novagen)1× buffer for KOD Hot Start DNA polymerase (Novagen)

1.5 mM MgSO₄

0.8 mM dNTPs (0.2 mM each)Cycling conditions: 95° C.^(3:00) (95° C.^(0:20)−70°C.^(0:40, 0:50, 1:00))×40 or 50 cycles

After the TSP cycles, the resulting products were diluted 100-fold inwater, and then underwent a further step of PCR containing 200 nM eachof the universal forward primer and 200 nM of the universal reverseprimer (cycling conditions: 95° C.^(3:00) (95° C.^(0:20)−70°C.^(0:40))×30 cycles). The resulting gene block was run on a 1% agarosegel at 100V for 1 hour 15 minutes (see FIG. 8). The expected productswere 1155 bp and 1308 bp respectively, and products of these sizes werevisualized on the gel.

SEQ ID No. 82: 10 sub-block gene blockCGACACTGCTCGATCCGCTCGCACCCCGCCTTGTTTAACTTTAAGAAGGAGCCCTTCCCCATGACAAGAACAAGTTTGCCTTTTCCAGACGGTTTCCTGTGGGGCGCAAGCACGGCGGCTCACCAGATTGAAGGTAATAATGTAAATAGTGATTGGTGGAGAAAAGAACATGACCCTGCTGCAAATATTGCAGAACCATCTTTGGATGCCTGTGACTCATATCACCGCTGGGAACAAGATATGGACCTGTTAGCAGAACTGGGCTTTACCGATTACCGCTTCTCCGTTGAATGGGCCCGTATTGAACCTGTGCCAGGTACATTTTCGCATGCTGAAACGGCACACTATCGTAGAATGGTTGATGGTGCTTTGGCAAGAGGCCTGCGCCCAATGGTCACCCTGCATCACTTTACTGTACCGCAGTGGTTCGAAGATTTGGGTGGCTGGACAGCCGATGGTGCCGCGGACCTGTTTGCACGTTACGTCGAACATTGTGCTCCGATTATCGGTAAAGATGTTAGACACGTGTGCACGATTAATGAACCTAACATGATCGCCGTAATGGCGGGCTTAGCTAAGACAGGCGATCAAGGTTTCCCACCGGCGGGTTTGCCTACGCCTGACGAAGAAACCACTCATGCTGTTATTGCTGCACATCACGCCGCGGTCAAAGCAGTACGTGCCATTGATCCGGACATCCAGGTCGGCTGGACCATCGCTAATCAAGTATATCAGGCATTACCTGGTGCCGAAGATGTTACTGCTGCATATCGTTACCCAAGAGAAGACGTGTTCATTGAAGCCGCTCGTGGCGATGACTGGATCGGCGTGCAATCTTACACACGCACGAAGATTGGTGCGGATGGCCCAATCCCGGCGCCTGAAGACGCTGAACGCACCCTGACTCAGTGGGAATATTACCCAGCTGCTGTTGGTCATGCTCTGCGTCACACAGCGGATGTCGCTGGCCCAGACATGCCGTTAATTGTAACCGAAAACGGTATCGCCACTGCGGATGACGCACGCCGTGTGGATTATTACACTGGTGCACTGGAAGCCGTTTCAGCCGCGTTAGAAGATGGTGTGAATATTCATGGCTATCTGGCGTGGAGCGCTTTGGATAACTATGAATGGGGTAGTTACAAACCGACTTTTGGCCTGATCGCAGTTGATCCTGTGACATTCGAAAGAACGGCCAAGCCGTCAGCAGTGTGGTTAGGTGAAATGGGTAGAACAAGACAGTTGCCAAGAGCGGAACGCGGGAAGGGTGGGCGCGCCGACCCGGATCGACGAGAGCAGC GCGACTGGSub-block sequences: SEQ ID No. 83:CGACACTGCTCGATCCGCTCGCACCCCGCCTTGTTTAACTTTAAGAAGGAGCCCTTCCCCATGACAAGAACAAGTTTGCCTTTTCCAGACGGTTTCCTGTGGGGCGCAAGCACGGCGGCTCACCAGATTGAAGGTAATAATGTAAATAGT GATTGGTGGAGSEQ ID No. 84: GCTCACCAGATTGAAGGTAATAATGTAAATAGTGATTGGTGGAGAAAAGAACATGACCCTGCTGCAAATATTGCAGAACCATCTTTGGATGCCTGTGACTCATATCACCGCTGGGAACAAGATATGGACCTGTTAGCAGAACTGGGCTTTACCGATTACCGCTTCTCCGTTG SEQ ID No. 85:GAACTGGGCTTTACCGATTACCGCTTCTCCGTTGAATGGGCCCGTATTGAACCTGTGCCAGGTACATTTTCGCATGCTGAAACGGCACACTATCGTAGAATGGTTGATGGTGCTTTGGCAAGAGGCCTGCGCCCAATG SEQ ID No. 86:CTTTGGCAAGAGGCCTGCGCCCAATGGTCACCCTGCATCACTTTACTGTACCGCAGTGGTTCGAAGATTTGGGTGGCTGGACAGCCGATGGTGCCGCGGACCTGTTTGCACGTTACGTCGAACATTGTGCTCCGATTATCGGTAAAGATG TTAGACACSEQ ID No. 87: GTCGAACATTGTGCTCCGATTATCGGTAAAGATGTTAGACACGTGTGCACGATTAATGAACCTAACATGATCGCCGTAATGGCGGGCTTAGCTAAGACAGGCGATCAAGGTTTCCCACCGGCGGGTTTGCCTACGCCTGACGAAGAAACC AC SEQ ID No. 88:GGGTTTGCCTACGCCTGACGAAGAAACCACTCATGCTGTTATTGCTGCACATCACGCCGCGGTCAAAGCAGTACGTGCCATTGATCCGGACATCCAGGTCGGCTGGACCATCGCTAATCAAGTATATCAGGCATTACCTGGTGCCGAAGA TGTTACTGSEQ ID No. 89: ATCAGGCATTACCTGGTGCCGAAGATGTTACTGCTGCATATCGTTACCCAAGAGAAGACGTGTTCATTGAAGCCGCTCGTGGCGATGACTGGATCGGCGTGCAATCTTACACACGCACGAAGATTGGTGCGGATGGCCCAATCCCGGCGCCTGAAGACGCTGAACGCACCCTGACTCAGTGGGAATATTACCC SEQ ID No. 90:CTGAACGCACCCTGACTCAGTGGGAATATTACCCAGCTGCTGTTGGTCATGCTCTGCGTCACACAGCGGATGTCGCTGGCCCAGACATGCCGTTAATTGTAACCGAAAACGGTATCGCCACTGCGGATGACGCACGCCGTGTGGATTATTACACTGGTGCACTGGAAGCCGTTTCAGCCGCGTTA SEQ ID No. 91:GCACTGGAAGCCGTTTCAGCCGCGTTAGAAGATGGTGTGAATATTCATGGCTATCTGGCGTGGAGCGCTTTGGATAACTATGAATGGGGTAGTTACAAACCGACTTTTGGCCTGATCGCAGTTGATCCTGTGACATTCGAAAGAACGGCC AAG SEQ ID No. 92:CAGTTGATCCTGTGACATTCGAAAGAACGGCCAAGCCGTCAGCAGTGTGGTTAGGTGAAATGGGTAGAACAAGACAGTTGCCAAGAGCGGAACGCGGGAAGGGTGGGCGCGCCGACCCGGATCGACGAGAGCAGCGCGACTGG

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

What is claimed is:
 1. A gene fragment library, said library comprising:a plurality of gene fragments wherein two or more of the gene fragmentsare comprised of a constant gene block_(a) of at least 100 bases and avariable gene block of at least 50 bases, wherein the constant geneblock sequence is identical for each of the gene fragments and thevariable gene block sequence varies.
 2. The gene fragment library ofclaim 1 wherein the gene fragments further comprise constant geneblock_(b).
 3. The gene fragment library of claim 2 wherein the variablegene block is flanked by constant gene block_(a) and constant geneblock_(b).
 4. The gene fragment library of claim 2 wherein the genefragments further comprise constant gene block_(n) wherein n representsa plurality of sets of constant gene blocks.
 5. The gene fragmentlibrary of claim 1 wherein the gene fragments further comprise variablegene block_(n) wherein n represents a plurality of sets of variable geneblocks
 6. The gene fragment library of claim 1 wherein the genefragments are at least 400 bases.
 7. The gene fragment library of claim1 wherein the gene fragments are at least 1000 bases.
 8. The genefragment library of claim 1 wherein the constant gene blocks or variablegene blocks located on terminal ends contain binding sites for forwardand reverse amplification primers.
 9. The gene fragment library of claim1 wherein the constant gene blocks and the variable gene blocks containoverlap regions for assembly into a gene fragment.
 10. The gene fragmentlibrary of claim 9 wherein the overlap regions contain a same sequenceof complementarity that allow for assembly with universal primers.